Protein phosphatase 1 (PP1) participates in the regulation of a wide variety of cellular functions by reversible protein phosphorylation and is one of the major protein phosphatases dephosphorylating serine and threonine residues in eukaryotes. The ability of PP1 to regulate diverse functions resides in the capacity of PP1 to interact with a variety of regulatory subunits that may target PP1 to specific subcellular locations, modulate its substrate specificity, and allow its activity to be responsive to extracellular signals [Hubbard and Cohen, Trends Biochem. Sci., 18:172-177 (1993)].
A 126 kDa glycogen binding subunit (G.sub.M or PPP1R3) targets PP1 to glycogen particles and to the sarcoplasmic reticulum in striated muscle [Tang et al., J. Biol. Chem., 266: 15782-15789 (1991); Chen et al., Diabetes, 43:1234-1241 (1994)]. The hormones insulin and adrenalin are thought to influence the activity of PP1 via the G.sub.M subunit. Phosphorylation of Ser-46 in human G.sub.M, response to insulin, enhances the rate at which at which PP1 dephosphorylates and activates glycogen synthase, causing an increase in glycogen synthesis [P. Dent et al, Nature, 348:302-308 (1990)]. In contrast, phosphorylation of Ser-65 in human G.sub.M by protein kinase A in response to .beta.-adrenergic agonists triggers dissociation of PP1 from G.sub.M, thus inhibiting PP1 from acting on glycogen synthase and phosphorylase and resulting in decreased glycogen synthesis and stimulation of glycogenolysis [Nakielny et al., Eur. J. Biochem., 199: 713-722 (1991)].
A distinct 33 kDa glycogen binding subunit (G.sub.L or PPP1R4), which is only 23% identical to the N-terminal portion of G.sub.M, targets PP1 to glycogen in liver [Moorhead et al., FEBS Lett., 362:101-105 (1994); Doherty et al., FEBS Lett., 375:284-289 (1995)]. The binding of G.sub.L modulates the activity of PP1, enhancing the rate at which it dephosphorylates and activates glycogen synthase and suppressing the rate at which it inactivates phosphorylase. Hormonal regulation of the activity of PP1 in liver is not known to occur through the phosphorylation of G.sub.L. Instead, the hormone glucagon (acting via cyclic AMP and protein kinase A) and a-adrenergic agonists (acting via Ca.sup.+2) increase the levels of phosphorylase a, which to binds G.sub.L and potently inhibits PP1 at nanomolar concentrations. This inhibition is though to be allosteric, since the K.sub.m for phosphorylase as a substrate of PP1 is in the micromolar range. Insulin acts by lowering the level of cyclic AMP in liver, thereby decreasing the level of phosphorylase a and relieving the inhibition of PP1-G.sub.L complex. Glycogen synthesis in liver is also stimulated by glucose, which binds to phosphorylase a, increasing the rate at which it is dephosphorylated.
Several other targeting subunits of PP1 have now been identified in mammals and these include the myosin binding targeting complex (comprising an M.sub.110 and M.sub.21 subunit) of smooth muscle, which enhances the dephosphorylation of myosin light chains by PP1 and is involved in the relaxation of smooth muscle [D. Alessi et al, Eur. J. Biochem., 210:1023-1035 (1992); Y. H. Chen et al, FEBS Lett., 356:51-55 (1994)]. A distinct myosin targetting subunit of PP1 is present in striated muscles [P. Dent et al, Eur. J. Biochem., 210:1037-1044 (1992)]. A p53 binding protein (53BP2) [N. R. Helps et al, FEBS Lett., 377:295-300 (1995)], a nuclear protein NIPP-1 [A. Van Eynde et al., J. Biol. Chem., 270:28068-28074 (1995)] and an RNA splicing factor PSF1 [K. Hirano et al, FEBS Lett., 389:191-194 (1996)] have been shown to bind to PP1. The retinoblastoma gene product [T. Durphee et al., Genes Dev., 7:555-569 (1993)], ribosomal proteins L5 [K. Hirano et al., J. Biol. Chem., 270:19786-19790 (1995)] and RIPP-1 [Buellens et al, Eur. J. Biochem., 239:183-189 (1996)] and a 110 kDa nuclear protein yet to be identified [I. Jagiello et al, J. Biol. Chem., 270:17257-17263 (1995)] are also reported to interact with PP1. The small cytosolic proteins, inhibitor-1, inhibitor-2 and DARPP-32 inhibit PP1 [P. Cohen, Annu. Rev. Biochem., 58:453-508 (1989)]. A complex between inhibitor-2 and PP1 has been isolated. More recently, inhibitor-2 has been shown to act like a molecular chaperon to fold PP1 into its native conformation [D. R. Alessi et al, Eur. J. Biochem., 213:1055-1066 (1993); C. MacKintosh et al, FEBS. Lett., (1996) in press]. A number of distinct PP1 targeting subunits have also been identified in yeast [M. J. R. Stark, Yeast (1996) in press].
Sites on the glycogen and myofibrillar targeting subunits which bind to PP1 have been localized [D. F. Johnson et al., Eur. J. Biochem., 239:317-325 (1996)] and a 13 residue peptide containing a RVXF motif common to many of the PP1 binding subunits has been crystallized as a complex with PP1 [M. Egloff et al., EMBO J. (1997) submitted].
Known PP1 subunits play a role in the regulation of glycogen metabolism and thus novel PP1 subunit proteins, agonists or antagonists thereof are anticipated to be beneficial in many diseases which involve resistance to the action of insulin on glycogen synthesis. Such disorders include, without limitation, diabetes mellitus, obesity, essential hypertension, dyslipidaemia and premature atherosclerosis, among others.
There, thus, exists a need in the art for a variety of PP1 binding proteins, antagonists and agonists thereof, as well as compositions and methods for the use of same.